Hepatitis C virus (HCV) infection is a causal agent of chronic liver disease in humans, afflicting more than 123 million people worldwide (approximately 2% of the human population). In many cases HCV infection becomes chronic, which can progress to liver cirrhosis and hepatocellular carcinoma. There is no vaccine available for HCV and the only treatment is pegylated interferon-? in combination with ribavirin, which leads to a sustained response in only 50% of patients. The high prevalence of infection, lack of HCV specific inhibitors, and poor response rate to the current treatment underscore the importance of new therapies. The HCV virion consists of a membrane enveloped nucleocapsid and two glycoproteins (E1 and E2), which are important for recognizing and entering a target host cell. As in the case of other enveloped viruses such as pestiviruses, alphaviruses, and other flaviviruses, HCV infection requires fusion between viral and host cell membrane before the viral genome can be released into the host cell cytosol. This critical step is thought to occur in the endosomal compartment, is triggered by low pH, and likely requires structural rearrangement of the glycoproteins. In addition to its role in membrane fusion, E2 is thought to be responsible for targeting since it has been shown to bind host cellular receptors CD81 and scavenger receptor class B type I (SR-BI). Despite the progress that has been made in understanding E2 function using virus-neutralizing antibodies and genetic approaches, more in-depth mechanistic dissection of E2 requires complementary biochemical, biophysical and molecular virology techniques. Progress has been hindered by the inability to make sufficient quantities of properly folded protein as E2 protein production is challenging due to its heavy glycosylation and presence of intramolecular disulfide bonds. Recently, our laboratory has devised and published a novel expression system in mammalian cells to produce eE2 (E2 ectodomain) protein that retains the functionality of E2 present on virions. Together with our comprehensive biochemical and biophysical characterization of eE2, we have established a foundation to better define the functional role of E2 in HCV infection. In this proposal, we will focus on assessing the role of E2 in triggering low-pH dependent membrane fusion and explore possible oligomeric and structural rearrangements necessary for this critical step in HCV infection. A multidisciplinary experimental plan that integrates biochemical, biophysical, genetic and molecular virology techniques will be used to: (1) define the physiological oligomeric form of E2 relevant to HCV infection; (2) characterize the role of E2 in sensing a decrease in pH, as encountered by the virus during endosomal acidification; and (3) determine the domain organization of E2 and possible rearrangements upon pH change. Results from the proposed studies will contribute substantially to our understanding of the mechanisms responsible for viral entry and have important implications for the development of novel therapies and vaccines to control HCV infection. Similar studies on the HIV glycoproteins have lead to the development of a new class of antiretroviral that block HIV entry. PUBLIC HEALTH RELEVANCE: Currently, 2% of the human population - approximately 123 million people worldwide - is chronically infected with hepatitis C virus (HCV), making virus transmission a major public health concern. There is no vaccine against HCV and the current drugs are not affective for many people. The goal of the proposed grant will provide a better understanding of how HCV recognizes and enter cells. This information will be valuable for designing new therapies or a vaccine against HCV.